Iodothyronine immunogens and antibodies

ABSTRACT

Immunogen conjugates comprising N-aminoalkyl derivatives of iodothyronines, e.g., thyroxine and its lower alkyl esters, coupled to an immunogenic carrier material, and antibodies raised against such immunogen conjugates.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of application Ser. No. 318,026, filedNov. 4, 1981, now U.S. Pat. No. 4,358,604 issued Nov. 9, 1982.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to immunogen conjugates comprising iodothyroninederivatives, particularly amino-functionalized derivatives, coupled toconventional immunogenic carrier materials, and antiiodothyronineantibodies prepared against such immunogen conjugates. Such antibodiesare useful in immunoassays for determining iodothyronines in biologicalfluids.

The iodothyronines have the following general formula: ##STR1## whereinβ¹ and β² are, independently, hydrogen or iodine. The iodothyronines ofclinical interest are listed in the following table:

    ______________________________________                                        Iodothyronine     β.sup.1                                                                             β.sup.2                                     ______________________________________                                        3,5,3'5'-tetraiodothyronine                                                                     iodine     iodine                                           (thyroxine; T-4)                                                              3,5,3'-triiodothyronine                                                                         iodine     hydrogen                                         (liothyronine; T-3)                                                           3,5',5'-triiodothyronine                                                                        hydrogen   iodine                                           ("reverse" T-3)                                                               3,3'-diiodothyronine                                                                            hydrogen   hydrogen                                         ______________________________________                                    

The quantitative determination of the concentration of the variousiodothyronines, particularly the hormones T-3 and T-4, in serum and ofthe degree of saturation of the iodothyronine binding sites on thecarrier protein thyroid binding globulin (TBG) are valuable aids in thediagnosis of thyroid disorders.

2. Brief Description of the Prior Art

Iodothyronines have been derivatized in many different ways in couplingthem to immunogenic carrier materials for the purpose of preparingimmunogen conjugates. Antibodies prepared against such conjugates areused in immunoassays for determining iodothyronines. Also,iodothyronines have been variously derivatized in order to couple adesired labeling moiety yielding a labeled conjugate useful in suchimmunoassays.

The commonly used technique for preparing iodothyronine immunogenconjugates involves coupling the iodothyronine directly through itsavailable amino and carboxyl groups to amide bond carboxyl and aminocounterpart groups on the carrier material in the presence ofcarbodiimide. A mixture of conjugates is obtained and consequently amixture of anti-iodothyronine antibodies are obtained. See Gharib et al,J. Clin. Endocrinol 33:509(1971).

U.S. Pat. No. 4,171,432 describes the coupling of a ribonucleotide toiodothyronines through the carboxyl group. U.S. Pat. No. 4,040,907describes the coupling of enzymes with iodothyronine derivativesderivatized at the phenolic hydroxyl group.

The preparation of aminoacyl thyroxine derivatives derivatized at theamino group of thyroxine is described in Endocrinol. 89:606-609(1971).

SUMMARY OF THE INVENTION

The present invention provides iodothyronine immunogen conjugates ofimmunogenic carriers coupled to iodothyronine derivatives bearing anaminofunctionalized arm at the amino group in iodothyronines and theiralkyl esters. The immunogens provided are of the general formula:##STR2## wherein Carrier is an immunogenic carrier material, R' is abond or a linking group, n is an integer from 2 through 12, p is on theaverage from 1 through about 50, R is hydrogen or alkyl containing 1-6carbon atoms, and β¹ and β² are, independently, hydrogen or iodine. Whenbridge group R' is a bond, the iodothyronine derivative is coupleddirectly to the carrier material, for example by amide linkages betweenthe amino group in the iodothyronine derivative and carboxyl groups inthe carrier, which in such case is usually a protein or polypeptide.When bridge group R' is other than a simple bond, it may comprise a widevariety of structures, for example, the residue of bifunctional linkingagents coupling the amino group in the iodothyronine derivative to aminogroups in the carrier, again usually a protein or polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 1a constitute a flow diagram of the synthetic routeillustrated in the Examples for preparing the iodothyronine derivativesthat are coupled to immunogenic carriers to form the immunogenconjugates of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The iodothyronine derivatives are generally prepared by the routeillustrated in the diagram of FIGS. 1 and 1a of the drawings. Anω-aminoalkylaldehyde diethyl acetal (1) is protected as theN-trifluoroacetate (2) by treatment with ethyl trifluoroacetate andtriethylamine in an appropriate solvent, e.g., ethanol. The resultingN-trifluoroacetylalkanal diacetal is hydrolyzed to the alkylaldehyde andreacted with an iodothyronine ethyl ester (3) and sodiumcyanoborohydride in an appropriate solvent, e.g., ethanol, to give theN-(ω-N-trifluoroacetylaminoalkyl) iodothyronine ethyl ester (4).Alkaline hydrolysis gives the novel N-(aminoalkyl)iodothyroninederivatives (6). Alternatively, the N-trifluoroacetyl group can beselectively removed by reflux in hydrochloric acid-saturated ethanol togive the dihydrochloride of (5) which has better organic solventsolubility properties.

The length of the linear alkyl portion of the N-aminoalkyl side arm canvary from 2 through 12 carbons by appropriate selection of theω-aminoalkyraldehyde diethyl acetal starting material. Similarly, thealkyl ester group, if present, will vary according to the selectediodothyronine alkyl ester starting material and can be linear orbranched and contain between 1 and 6 carbon atoms, e.g., methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, and so forth.

The N-(aminoalkyl)iodothyronine ester derivatives (5) are converted tothe corresponding acid derivatives (6) by treatment with base.

The ester and acid iodothyronine derivatives can be coupled byconventional peptide condensation reactions to carboxyl group-containingimmunogenic carrier materials to yield immunogen conjugates of theformula: ##STR3## wherein Carrier (CO)-- represents the carrier materialbound through a carboxyl group, p is on the average from 1 through thenumber of available carboxyl groups on the carrier, and n, R, β¹ and β²are as defined above. The peptide condensation reactions available forperforming the direct coupling of the iodothyronine derivative to acarboxyl group-containing carrier are well known and include, withoutlimitation, the carbodiimide reaction [Aherne et al, Brit. J. Clin.Pharm. 3:56(1976) and Science 144:1344(1974)], the mixed anhydridereaction [Erlanger et al, Methods in Immunology and Immunochemistry, ed.Williams and Chase, Academic Press (New York 1967) p. 149], and the acidazide and active ester reactions [Kopple, Peptides and Amino Acids, W.A. Benjamin, Inc. (New York 1966) ]. See also Clin. Chem. 22:726(1976).

Alternatively, the ester and acid iodothyronine derivatives can becoupled through the use of linking reagents that form a bond at one endwith the amino group in the iodothyronine derivative and a bond at theother end with an appropriate functional group present in the carriermaterial. For example, bifunctional coupling reagents are well known forcoupling amine derivatives to amine macromolecules, includingbis-imidates, bis-isocyanates, and glutaraldehyde [Immunochem.6:53(1969)]. Other useful coupling reactions are thoroughly discussed inthe literature, for instance in the above-mentioned Kopple monograph;Lowe and Dean, Affinity Chromatography, John Wiley & Sons (New York1974); Means and Feeney, Chemical Modification of Proteins, Holden-Day(San Francisco 1971); and Glazer et al, Chemical Modification ofProteins, Elsevier (New York 1975).

Coupling of the iodothyronine derivative to a protein or polypeptidecarrier through the use of the bis-imidate bifunctional reagents is apreferred method. The resulting immunogen conjugates will comprise alinking group R' of the formula: ##STR4## wherein m is an integer from 1through 10 and the carrier material is coupled through an amino group.The bis-imidate coupling reagents will generally be of the formula:##STR5## wherein m is as defined above and R¹ and R², which may be thesame or different but which more usually are the same, are alkyl,preferably lower alkyl (i.e., having 1-4 carbon atoms) such as methyl,ethyl, n-propyl, iso-propyl, and so forth. Particularly preferredbis-imidates are the dimethyl alkylimidates, especially dimethyladipimidate. The bis-imidates are generally available from commercialsources or may be prepared by published methods by those skilled in theart [Hunter and Ludwig, J. Am. Chem. Soc. 84:3491(1962)].

The bis-imidates will normally be provided in a suitable salt form whichupon dissolution in the aqueous reaction media generates the positivelycharged bis-imidate species. Correspondingly, isolation of the immunogenconjugate from aqueous media such as by solvent evaporation orprecipitation yields salts forms of the bis-imidates wherein the counteranions to the protonated imino groups are taken from available anions inthe media.

The coupling reaction is allowed to proceed in aqueous solution undermild conditions, e.g., at a pH between about 7 and about 10, moreusually between 8 and 9, and at temperatures between about 0° C. andabout 40° C., more usually between 20° C. and 30° C. Usually, theamino-functionalized iodothyronine derivative, the bis-imidate, and thedesired protein or polypeptide carrier material are added in sequence,with a short incubation period for reaction between the iodothyroninederivative and the bis-imidate of between 1 and 30 minutes, followed byaddition of the protein or polypeptide and a second incubation periodlasting between 10 minutes and 4 hours.

The quantity p in the above formulas represents the number ofiodothyronine moieties that are conjugated to the carrier, i.e., theepitopic density of the immunogen, and in the usual situation will be onthe average from 1 to about 50, more normally from 1 to about 20.Optimal epitopic densities, considering the ease and reproducibility ofsynthesis of the immunogen and antibody response, fall between about 2and about 15, more usually between 4 and 10.

The immunogenic carrier material can be selected from any of thoseconventionally known having available functional groups for coupling tothe iodothyronine amino-derivatives. In most cases, the carrier will bea protein or polypeptide, although other materials such ascarbohydrates, polysaccharides, lipopolysaccharides, nucleic acids andthe like of sufficient size and immunogenicity can likewise be used. Forthe most part, immunogenic proteins and polypeptides will have molecularweights between 4,000 and 10,000,000, preferably greater than 15,000,and more usually greater than 50,000. Generally, proteins taken from oneanimal species will be immunogenic when introduced into the blood streamof another species. Particularly useful proteins are albumins,globulins, enzymes, hemocyanins, glutelins, proteins having significantnonproteinaceous constituents, e.g., glycoproteins, and the like. Thealbumins and globulins of molecular weight between 30,000 and 200,000are particularly preferred. Further reference for the state-of-the-artconcerning conventional immunogenic carrier materials and techniques forcoupling haptens thereto may be had to the following: Parker,Radioimmunoassay of Biologically Active Compounds, Prentice-Hall(Englewood Cliffs, New Jersey U.S.A., 1976); Butler, J. Immunol. Meth.7:1-24(1974); Weinryb and Shroff, Drug Metab. Rev. 10:271-283(1975);Broughton and Strong, Clin. Chem. 22:726-732(1976); and Playfair et al,Br. Med. Bull. 30:24-31(1974).

Preparation of specific antibodies using the present immunogenconjugates may follow any conventional technique. Numerous texts areavailable describing the fundamental aspects of inducing antibodyformation; for example reference may be made to Parker, Radioimmunoassayof Biologically Active Compounds, Prentice-Hall (Englewood Cliffs, NewJersey U.S.A., 1976). In the usual case, a host animal such as a rabbit,goat, mouse, guinea pig, or horse is injected at one or more of avariety of sites with the immunogen conjugate, normally in mixture withan adjuvant. Further injections are made at the same site or differentsites at regular or irregular intervals thereafter with bleedings beingtaken to assess antibody titer until it is determined that optimal titerhas been reached. The host animal is bled to yield a suitable volume ofspecific antiserum. Where desirable, purification steps may be taken toremove undesired material such as nonspecific antibodies before theantiserum is considered suitable for use in performing actual assays.

The antibodies can also be obtained by somatic cell hybridizationtechniques, such antibodies being commonly referred to as monoclonalantibodies. Reviews of such monoclonal antibody techniques are found inLymphocyte Hybridomas, ed. Melchers et al, Springer-Verlag (New York1978), Nature 266:495 (1977), and Science 208:692 (1980).

The present invention will now be illustrated, but is not intended to belimited, by the following examples.

EXAMPLE 1 Preparation of N-aminoalkyl iodothyronine derivatives andtheir alkyl esters

The reaction sequence for the preparation of these iodothyroninederivatives is shown in the diagram in FIGS. 1 and 1a of the drawings.

4-N-(Trifluoroacetyl)aminobutyraldehyde Diethylacetal (2), n=4

To a mixture of 17.74 grams (g) of 90% 4-aminobutyraldehydediethylacetal (1) [0.1 moles (mol)] and 22.1 milliliters (ml)triethylamine (0.16 mol) in 100 ml dry ethanol at 0° C. under argon gaswas added dropwise 21.3 g ethyl trifluoroacetate (0.16 mol) over 15minutes. The mixture was allowed to warm to room temperature overnight.The reaction volume was concentrated in vacuo (bath temperature <40°C.), dissolved in ether, and washed with water and brine. Drying (Na₂SO₄), filtration, and evaporation of solvent in vacuo gave 25.3 g ofbrown oil. Fractional distillation at 104°-105° C. (0.01 mm) providedthe product (2), n=4, as a colorless oil (96%, 24.6 g).

Analysis: Calculated for C₁₀ H₁₈ NF₃ O₃ (MW 257.26): C, 46.7; H, 6.8; N,5.5. Found: C, 46.3; H, 7.0; N, 5.3.

PMR (60 mHz, CDCl₃): δ1.2 (t, J=7 Hz,6H); 4.3 (m,4H); 3.2-4.0 (m,6H);4.52 (t,J=5 Hz,1H); 7.3 (bs,1H).

IR(neat): 1705 cm⁻¹.

N-[4-N-(Trifluoroacetyl)aminobutyl]thyroxine Ethyl Ester (4), n=4, β¹=β² =I

4-N-(Trifluoroacetyl)aminobutyraldehyde diethylacetal (2), n=4, [3.097g, 12 millimoles (mmol)] was stirred in a 36 ml mixture oftetrahydrofuran/acetic acid/water (1:1:1) for 48 hours under argon gas.The mixture was evaporated in vacuo to an oil which was added to 10.09 g(12 mmol) thyroxine ethyl ester hydrochloride (3), β¹ =β² =I, in 275 mlabsolute ethanol under argon gas. Sodium cyanoborohydride [422milligrams (mg), 6.8 mmol] was added to the mixture, which was allowedto stir 24 hours at room temperature. The mixture was filtered andconcentrated in vacuo to a foam, which was dissolved in 40 ml 20% ethylacetate-dichloromethane, filtered, and allowed to stand overnight. Whitecrystals of product appeared which were collected, washed withdichloromethane, and dried to yield 4.32 g (37%), mp 202°-204° C.

Analysis: Calculated for C₂₃ H₂₂ N₂ F₃ I₄ O₅.2H₂ O (MW 1008.12): C,27.40; H, 2.70; N, 2.78. Found: C, 27.36; H, 2.45; N, 2.77.

PMR (60 mHZ, DMSO-d₆): δ1.13(t,J=3 Hz, 3H); 1.67 (m,4H); 2.7-3.9 (m,6H);4.2 (m+superimposed q, J=7 Hz,3H); 7.12 (s,2H), 7.92 (s,2H); 9.65(m,3H).

IR (KCl): 1720, 1745 cm⁻¹.

The mother liquor was concentrated in vacuo, dissolved in 10 ml ethylacetate, and chromatographed on a Waters Prep 500® HPLC (WatersAssociates, Inc., Milford, MA) using one Prep-Pak-500® 5.7 ID×30 cmsilica cartridge (Waters Associates, Inc., Milford, MA) as a stationaryphase and 20% ethyl acetatedichloromethane as the mobile phase.

First eluted from the column was an oil which was precipitated as awhite powder (1.1 g) from dichloromethane-hexane, mp 64°-68° C.decomposed. It was identified asN,N-bis-[4-N-(trifluoroacetyl)aminobutyl]thyroxine ethyl ester on thebasis of the following analytical data (7% yield).

Analysis: Calculated for C₂₉ H₃₁ N₃ F₆ I₄ O₆.H₂ O (MW 1157.24): C,30.10; H, 2.87; N, 3.63. Found: C, 30.01; H, 2.45; N, 4.13.

PMR (60 mHZ, DMSO-d₆): δ1.15 (m+bt, J=7 Hz,7H); 1.65 (m,4H); 2.9 (m,3H);3.2 (m,4H); 3.7 (m,3H); 4.1 (m+q, J=7 Hz, 3H); 5.73 (m,2H); 7.13 (s,2H);7.88 (s,2H); 9.4 (m,1H).

IR (KCl): 1720 cm⁻¹.

Next obtained from the column was an oil, which was precipitated as3.886 g of a white powder from dichloromethane-hexane. Spectral datashowed it to be identical with the initially formed crystals. The totalyield of product (4), n=4, β¹ =β² =I, was 8.23 g (71%), mp 105°-106°.

Analysis: Calculated for C₂₃ H₂₃ N₂ O₅ I₄ F₃ (MW 972.09): C, 28.41; H,2.39; N, 2.88. Found: C, 28.33; H, 2.40; N, 2.78.

N-(4-Aminobutyl)thyroxine Ethyl Ester Bishydrochloride (5), n=4, β¹ =β²=I.

N-[4-N-(Trifluoroacetyl)aminobutyl]thyroxine ethyl ester (4), n=4, β¹=β² =I, (2.5 g, 2.6 mmol) was dissolved in 100 ml anhydrous ethanol. Thesolution was heated to reflux while treated with gaseous hydrochloricacid for 7 hours. The mixture was concentrated in vacuo and the residuetwice precipitated from ethanol-ether to give 2.115 g of an off-whitepowder (79% yield) which analyzed as the bishydrochloride diethanolate(5), n=4, β¹ =β² =I.

Analysis: Calculated for C₂₁ H₂₄ N₂ I₄ O₄.2HCl.2(C₂ H₆ O) (MW 1041.13):C, 28.84; H, 3.68; N, 2.69. Found: C, 29.32; H, 3.28; N, 2.91.

PMR (90 mHZ, DMSO-d₆): δ1.13 (t, J=7 Hz); 1.77 (m); 3.29 (m); 3.66 (m);4.67 (m); 7.17 (s); 7.88 (s); 8.79 (m); 10.0 (m); 10.6 (m).

IR (KCl): 1735, 2970 cm⁻¹.

N-(4-Aminobutyl)thyroxine (6), n=4, β¹ =β² =I

N-[4-N-(Trifluoroacetyl)aminobutyl]thyroxine ethyl ester (4), n=4, β¹=β² =I, 2.0 g (2.06 mmol) was dissolved in 20 ml tetrahydrofuran underargon gas and treated with 4.1 ml of a 2 N sodium hydroxide solution.The mixture was stirred overnight at 50° C. The reaction mixture wasthen preadsorbed onto a small amount of Silicar CC-7 (silicic acid;Mallinkrodt, Inc., Paris, KY) and applied to the top of a column of 175g Silicar CC-7 packed with the lower phase of a 2:1:1chloroform-methanol-ammonium hydroxide mixture. The column wassequentially eluted with 3 liters each of the lower phase of a 2:1:1 and1:1:1 chloroform-methanol-ammonium hydroxide mixture. The off-whitepowder obtained was dissolved in ethanol and 5 ml 2 N sodium hydroxide,treated with Norit filtered through celite, and was precipitated withacetic acid. Obtained was 1.5 g (88% yield) of the conjugate (6), β¹=β.sup. 2 =I, as amorphous powder, mp 166° decomposed.

Analysis: Calculated for C₁₉ H₂₀ I₄ O₃.H₂ O (MW 850.05): C, 26.85; H,2.61; N, 3.30. Found: C, 26.47; H, 2.51; N, 3.29.

PMR (100 mHz, CD₃ OD+Na°): δ1.48 (m); 2.64 (m); 2.82 (dd,J=6 Hz, 8 Hz);3.26 (t,J=6 Hz); 7.04 (s); 7.86 (s).

IR (KCl): 1640, 1435, and 1505 cm⁻¹.

The hydrochloride was prepared by stirring 100 mg of the product in 6 mlof 6 N hydrochloric acid for 2 hours at reflux under argon gas.Filtration and drying at 55° C. (0.1 mm Hg) for 17 hours gave 82 mg ofthe product (79% yield).

Analysis: Calculated for C₁₉ H₂₀ N₂ I₄ O₃.HCl.H₂ O (MW 886.50): C,25.74; H, 2.62; N, 3.16. Found: C, 25.87; H, 2.34; N, 3.17.

EXAMPLE 2 Preparation of antibodies to thyroxine using bis-imidatecoupled immunogen

Eighty-three milligrams of N-(4-aminobutyl)thyroxine (Example 1) wasadded to 3.0 ml of 0.1 M sodium carbonate, and 35 μl of 0.1 N sodiumhydroxide was added. Dimethyladipimidate dihydrochloride (90 mg) wasadded and a precipitate formed. The precipitate dissolved upon additionof 70 μl of 10 N sodium hydroxide. When this reaction mixture had stoodat room temperature for 3 to 5 minutes, it was added dropwise to astirred solution containing 200 mg of bovine serum albumin in 50 ml of0.2 N sodium hydroxide. This mixture was allowed to stand at roomtemperature for 2.5 hours and then the pH was adjusted to 7.0 with 5 Nhydrochloric acid. The precipitate which formed was removed bycentrifugation, and the supernatant was concentrated to 5-7 ml bypressure dialysis.

The concentrate was chromatographed on a 2.5×45 cm column of SephadexG25 (coarse) equilibrated with 0.1 M sodium phosphate, pH 7.0 containing0.02% sodium azide. About 12 ml fractions were collected and fractions9-12 were pooled.

Two-tenths milliliter of the pool was diluted into 0.8 ml of 0.1 Nsodium hydroxide, and the optical absorption spectrum from 260 to 360nanometers (nm) was recorded. An absorption maximum occurred at 328 nm.The absorbances at 280 and 328 nm were used to estimate an incorporationof 6.8 moles of N-(4-aminobutyl)thyroxine per mole of bovine serumalbumin.

The N-(4-aminobutyl)thyroxine bovine serum albumin conjugate pool wasdiluted to give 0.6 mg conjugate per ml. For the initial immunization,this solution was blended with an equal volume of Freund's completeadjuvant. One-tenth milliliter of the mixture was injectedsubcutaneously into each foot pad of a rabbit and 0.6 ml was injectedsubcutaneously on the back of the rabbit. Booster immunizations wereadministered on the back 3, 7, 11, and 15 weeks later. Theseimmunizations employed Freunds incomplete adjuvant. Blood was drawn onthe 16th week and serum was collected by centrifugation.

Antibody binding reaction mixtures were prepared for titrating thethyroxine antibodies by combining reagents in the order presented inTable A. The ¹²⁵ I labeled thyroxine (Tetramat, Abbott Laboratories,Inc., Chicago, IL) gave about 630,000 counts per minute (cpm) per mlwhen measured on a Gammacord® crystal scintillation instrument (AmesDivision, Miles Laboratories, Inc., Elkhart, IN). All of the reagentswere prepared in 0.1 M sodium phosphate buffer, pH 7.0. The antibody wasallowed to incubate with the ¹²⁵ I-thyroxine for 2 to 3 hours at roomtemperature before the 50% polyethylene glycol was added. After thepolyethyleneglycol was added and mixed, insoluble proteins weresedimented by centrifugation and the supernatant was decanted. Theradioactivity in the pellet was measured.

                  TABLE A                                                         ______________________________________                                        0.1M                              50%                                         Sodium                                                                              Normal                      Poly-                                       Phos- Rabbit   Antiserum          Ethyl-                                      phate Immuno-  to         .sup.125 I-                                                                           ene   Radio-                                pH 7.0                                                                              globulin Thyroxine  Thyroxine                                                                             glycol                                                                              activity                              (μl)                                                                             (μl)  (5 μl/ml) (μl)                                                                     (μl) (μl)                                                                             (cpm)                                 ______________________________________                                        480   20       --         100     400     4600                                470   20       10         100     400   39,100                                460   20       20         100     400   41,600                                380   20       100        100     400   43,500                                ______________________________________                                    

EXAMPLE 3 Thyroxine Radioimmunoassay

Competitive binding reaction mixture were prepared with various levelsof thyroxine by combining reagents in the order presented in Table B(the antiserum used having been raised against the bis-imidate coupledimmunogen of Example 2). The ¹²⁵ I-thyroxine solution gave about 450,000counts per minute (cpm) per ml. The reactions were allowed to incubateat room temperature for 2 to 3 hours before the 50% polyethyleneglycolwas added. The insoluble proteins were collected as outlined above andthe radioactivity was measured.

The results show that as the level of thyroxine increased, the amount¹²⁵ I-thyroxine bound to antibody decreased.

                                      TABLE B                                     __________________________________________________________________________                                Antiserum                                         0.1M Sodium                 to                                                Phosphate                                                                            Normal Rabbit                                                                          .sup.125 I-                                                                         Thyroxine                                                                           Thyroxine                                                                           50% Poly-                                   pH 7.0 Immunoglobulin                                                                         Thyroxine                                                                           (5 μM)                                                                           (5 μl/ml)                                                                        Ethyleneglycol                                                                        Radioactivity                       (μl)                                                                              (μl)  (μl)                                                                             (μl)                                                                             (μl)                                                                             (μl) cpm                                 __________________________________________________________________________    480    20       100   --    --    400      4,700                              380    20       100   --    100   400     22,000                              380    20       100   1     100   400     13,100                              380    20       100   2     100   400     10,600                              380    20       100   3     100   400      6,400                              330    20       100   50    100   400      3,900                              __________________________________________________________________________

EXAMPLE 4 Nonradioisotopic immunoassay for thyroxine

Reaction mixtures were formed by combining NADPH, buffer, thyroxineantibody (Example 2) and varying levels of thyroxine in the amounts andconcentrations indicated in Table C and incubated at room temperaturefor a minimum of 30 seconds. Then 100 μl of 0.16 μMmethotrexate-aminobutyl thyroxine conjugate [prepared fromN-(4-aminobutyl)thyroxine ethyl ester bishydrochloride (5), n=4, β¹ =β²=I, supra, as described in U.S. Patent Application Ser. No. 318,028,filed on Nov. 4, 1981 and assigned to the present assignee] was addedand the mixture was incubated at room temperature for 2 minutes.Following the second incubation, dihydrofolate reductase was added andthe mixture incubated an additional 5 minutes at 37° C. The reaction wasinitiated by adding dihydrofolate and an initial absorbance at 340 nmwas recorded. The reaction was allowed to proceed for 20 minutes at 37°C. and a final reading of absorbance recorded. The results in Table Cshow that the enzyme activity (ΔA) decreased as the thyroxine levelincreased.

                                      TABLE C                                     __________________________________________________________________________                   Antibody                                                                            Methotrexate-                                            0.1M Sodium    to    Aminobutyl- Dihydrofolate                                Phosphate Buffer                                                                       Thyroxine                                                                           Thyroxine                                                                           Thyroxine                                                                            NADPH                                                                              Reductase                                                                            Dihydrofolate                         pH 6.0   1 × 10.sup.-7 M                                                               1:4 Dil                                                                             0.16 μM                                                                           2.1 mM                                                                             0.04 U/ml                                                                            0.5 mM ΔA.sub.340/              (μl)  (μl)                                                                             (μl)                                                                             (μl)                                                                              (μl)                                                                            (μl)                                                                              (μl)                                                                              20 Min                         __________________________________________________________________________    1800     --    --    --     95   50     55     0.620                          1700     --    --    100    95   50     55     0.458                          1670     --    30    100    95   50     55     0.645                          1650      20   30    100    95   50     55     0.637                          1630      40   30    100    95   50     55     0.610                          1590      80   30    100    95   50     55     0.595                          1550     120   30    100    95   50     55     0.595                          1510     160   30    100    95   50     55     0.585                          1470     200   30    100    95   50     55     0.584                          __________________________________________________________________________

EXAMPLE 5 Preparation of antibodies to thyroxine using directly coupledimmunogen

An immunogen was prepared by coupling the primary amino group ofN-(4-aminobutyl)thyroxine to the carboxyl groups of bovine serumalbumin. The protein (151 mg dissolved in 3 ml water) was cooled in anice bath. N-(4-aminobutyl)thyroxine ethyl ester (39 mg) was dissolved in5 ml of water and added to the protein solution. The pH was 4.3.1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (202 mg dissolved in 2 mlof water) was cooled and added dropwise to the albumin,N-(4-aminobutyl)thyroxine solution. The pH was 5.1 and was adjusted to4.6 with 0.1 M HCl.

The reaction was allowed to proceed for 5 hours at 4° C. Then thereaction mixture was applied to a 3×50 cm column of Sephadex G-50 (fine)equilibrated with 50 mM sodium acetate buffer, pH 5.0. Four milliliterfractions were collected and the first eluted peak of material withabsorbance at 280 nm was pooled and dialyzed against 0.15 M sodiumchloride.

This immunogen had 6.0 moles of thyroxine residues per mole of bovineserum albumin.

The ester function of the thyroxine residues was hydrolyzed with alkali.Twenty-five milliliters of the dialyzed immunogen was combined with 0.25ml 1 M sodium hydroxide which gave a pH of 12. This mixture was allowedto stand at room temperature for 18 hours. Then the solution wasadjusted to pH 7.5 with 1 M HCl.

Four milliliters of immunogen (1 mg/ml) was combined with 10 ml ofFreunds complete adjuvant and 2 ml of saline. Rabbits were immunizedsubcutaneously each with 2 ml of this mixture. Three weeks later theywere reimmunized with the same mixture prepared with incomplete Freundsadjuvant. The booster immunizations were repeated every 4 weeks. Testbleedings were taken one week after the boosters. Antiserum withsuitable titers were obtained by 4 months after the initialimmunization.

EXAMPLE 6 Titration of thyroxine antibodies

The following reagents were prepared.

Buffer--0.1 M sodium phosphate, pH 7.0, containing 0.1% (w/v) bovineserum albumin and 0.1% (w/v) sodium azide.

Thyroxine--200 ng thyroxine/ml of the 0.1 M sodium phosphate buffer, pH7.0.

Antiserum to Thyroxine--The antiserum (from Example 5) was diluted into0.1 M sodium phosphate buffer, pH 7.0, such that 20 ml aliquots added tothe assay gave antiserum levels indicated in the table below.

Composite Reagent--0.1 M sodium phosphate buffer, pH 7.0, containing0.1% (w/v) sodium azide, 0.125 M glucose, 2.5 mM2-hydroxy-3,5-dichlorobenzene sulfonate, 1.25% (w/v) bovine serumalbumin and 23.8 μg horseradish peroxidase/ml.

Apoglucose Oxidase Reagent--0.1 M sodium phosphate buffer, pH 7.0, 6.0μM apoglucose oxidase binding sites (see U.S. Pat. No. 4,268,631), 12 mM4-amino antipyrine, 0.1% (w/v) sodium azide, 400 μl antiserum to glucoseoxidase/ml 30% (v/v) glycerol.

FAD-Thyroxine--0.1 M sodium phosphate buffer, pH 7.0, containing 40 nMflavin adenine dinucleotide conjugate. The conjugate was prepared asdescribed in U.S. Pat. No. 4,171,432.

Two series of cuvettes (12 each) were designated A and B. Buffer (0.23ml) was added to sets A and B. Fifty microliter of the Thyroxine Reagentwas added to each cuvette in set B.

Antiserum was added to cuvettes in each set such that the levels givenin the table below were achieved. The cuvettes were allowed to stand atambient temperature for at least 5 minutes.

One and six-tenths mililiters of Composite Reagent was added to eachcuvette. Then 50 μl of FAD-thyroxine was added followed immediately by50 μl of Apoglucose Oxidase Reagent. The reactions were incubated at 37°C. for 15 minutes and then the absorbance at 520 nM was recorded.

The results were as follows:

    ______________________________________                                        Antiserum        Absorbances at 520 nM                                        (μl/assay)    set A  set B                                                 ______________________________________                                        0                1.06   1.10                                                  2.5              0.614  0.778                                                 5.0              0.394  0.530                                                 7.5              0.375  0.445                                                 10.0             0.326  0.335                                                 20.0             0.107  0.110                                                 ______________________________________                                    

The results from set A show that the glucose oxidase activity decreasedas the antibody level increased indicating that the antibody inactivatedthe flavin adenine dinucleotide thyroxine conjugate. In the presence ofthyroxine, set B, the absorbances were higher indicating that thyroxineand the conjugate competed for antibody binding sites.

What is claimed is:
 1. An iodothyronine immunogen conjugate of theformula: ##STR6## wherein Carrier is an immunogenic carrier material, R'is a bond or a linking group, n is an integer from 2 through 12, p is onthe average from 1 through about 50, R is hydrogen or alkyl containing1-6 carbon atoms, and β¹ and β² are, independently, hydrogen or iodine.2. The conjugate of claim 1 wherein R' is a bond or a chain comprisingbetween 1 and 20 atoms excluding hydrogen.
 3. The conjugate of claim 1wherein R' is a 15 bond and the carrier material is coupled throughcarboxyl groups of the carrier material.
 4. The conjugate of claim 1wherein R' is a linking group of the formula: ##STR7## wherein m is aninteger from 1 through 10 and the carrier material is coupled throughamino groups.
 5. The conjugate of claim 1 wherein R is hydrogen.
 6. Theconjugate of claim 1 wherein R is ethyl.
 7. The conjugate of claim 5 or6 wherein n=4.
 8. The conjugate of any one of claims 1-4 wherein β¹ andβ² are both iodine.
 9. An iodothyronine immunogen conjugate of theformula: ##STR8## wherein Carrier (CO)--represents an immunogenicprotein or polypeptide carrier material bound through a carboxyl group,n is an integer from 2 through 12, p is on the average from 1 throughthe number of available carboxyl groups on said carrier material, R ishydrogen or alkyl containing 1-6 carbon atoms, and β¹ and β² are,indpendently, hydrogen or iodine.
 10. The conjugate of claim 9 wherein Ris hydrogen.
 11. The conjugate of claim 9 wherein R is alkyl containing1-6 carbon atoms.
 12. The conjugate of claim 11 wherein R is ethyl. 13.The conjugate of any one of claims 9-12 wherein n is
 4. 14. Theconjugate of any one of claims 9-12 wherein β¹ and β² are both iodine.15. The conjugate of claim 9 wherein p is on the average from 1 through20.
 16. The conjugate of claim 9 wherein said carrier material is analbumin.
 17. An antibody prepared against the conjugate of claim
 1. 18.An antibody prepared against the conjugate of claim
 3. 19. An antibodyprepared against the conjugate of claim
 4. 20. An antibody preparedagainst the conjugate of claim
 8. 21. An antibody prepared against theconjugate of claim
 9. 22. An antibody prepared against the conjugate ofclaim
 13. 23. An antibody prepared against the conjugate of claim 14.